Assays for Determining Anticoagulant Cofactor Activity

ABSTRACT

Methods are disclosed for determining, in a sample derived from a human, the functional activity of a component of the human blood coagulation system, which activity can be correlated to conversion of a substrate specific for activated Protein C (APC), by measuring in an assay medium containing the sample and a substrate for APC, the conversion of the substrate by APC and correlating the conversion to the functional activity of the component. When the component is anticoagulant Factor V, at least one of exogenous APC, Protein S or an inhibitor of Protein S activity is added to the medium. When the component is Protein C, APC, or Protein S, exogenous anticoagulant Factor V or an inhibitor of anticoagulant activity of Factor V is added to the medium. Methods are also disclosed for diagnosing a blood coagulation/anticoagulation disorder or for determining a predisposition thereto in a human by determining anticoagulant Factor V activity in an assay medium containing a sample derived from the human.

RELATED APPLICATIONS

Priority is claimed to U.S. patent application Ser. No. 08/500,917,which is a national phase application of International ApplicationPCT/SE94/00070, filed Jan. 28, 1994, which claims priority to SwedishApplication No. 9300300-2, filed Jan. 29, 1993, and Swedish ApplicationNo. 9302457-8, filed Jul. 20, 1993, the disclosures of which are herebyincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention is related to the diagnosis and treatment of bloodcoagulation disorders. In particular, the present invention is generallyrelated to a novel anticoagulant cofactor activity involved in the humanblood coagulation system and possibly also involved in the bloodcoagulation system of some other mammalian species.

BACKGROUND OF THE INVENTION

Blood coagulation is a complex system involving a large number ofproteins that function in concert with platelets to yield hemostasis.The coagulation system is strictly regulated by a series ofanticoagulant proteins present in plasma and on the surface ofendothelial blood cells (Esmon, J. Biol. Chem. 264 (1989) 4743-4746;Bauer, Sem. Hematol. 28 (1991)10-18; and Rapaport, Blood 73 (1989)35965). Under physiological conditions, pro- and anti-coagulantmechanisms are delicately balanced to provide hemostasis andcoagulation. Disturbances in this balance result in either bleeding orthromboembolic disorders.

The present invention is related to a novel activity involved in aphysiologically important anticoagulant system associated with Protein Cand Protein S that has been elucidated in recent years and is shown aspart of the blood coagulation interactions illustrated in FIG. 5.

In the above mentioned anticoagulant system, Protein C, a vitaminK-dependent plasma protein, is a key component that, after activation toActivated Protein C (APC) on endothelial cells by thethrombin/thrombomodulin complex, selectively degrades the coagulationFactors Va and VIIIa, i.e., the activated forms of the coagulationFactors V and VIII, respectively. (Esmon, supra; Stenflo, in Protein Cand Related Proteins, ed. Bertina (Churchill Livingstone Longham Group,UK) (1988) 21-54; Mann et al., Ann. Rev. Biochem. 57 (1988) 915-956; andKane et al., Blood 71 (1988) 539-55).

The activity of APC is influenced by another vitamin K-dependent plasmaprotein, designated Protein S, which functions as a cofactor to APC inthe degradation of Factors Va and VIIIa (Esmon, supra; Stenflo, supra;and Dahlbäck, Thromb. Haemostas. 66 (1991) 49-61).

The above mentioned Factors Va and VIIIa are phospholipid-boundcofactors involved in the activation of Factor X and prothrombin,respectively, and are, thus, indirectly involved in the conversion offibrinogen to fibrin, i.e., in clot formation. Accordingly, the rate ofthe coagulation reaction is dependent on the balance between theactivation of Factors VIII and V and the degradation of their activatedforms, the unactivated Factors VIII and V being poor substrates for APC.

Disturbances in the blood coagulation system are frequently manifestedas serious and often life-threatening conditions, and knowledge aboutthe underlying causes for the disturbances is often crucial in order toenable diagnosis and/or successful therapy of a manifested disease orthe screening of individuals having a predisposition for a bloodcoagulation disease. For instance, therapeutic use of purified Protein Chas been developed as a result of the discovery of Protein C deficiencyassociated with thrombophilia.

Thrombophilia can be defined as a tendency towards early-onset venousthomboembolic disease in adults in the absence of known risk factors.Although abnormalities have been determined for some thrombophilicpatients, in the majority of such cases no laboratory test abnormalitieswere identified.

The present invention is related to a new defect in anticoagulantresponse to activated Protein C, called APC-resistance, which has beenshown to be inherited and associated with familial thrombophilia.

In a few cases thrombophilia has been associated with hypotheticalfactors, such as an anti-Protein C antibody (Mitchell et al., NewEngland Journal of Medicine, 1987, Vol. 316, 1638-1642), ananti-cardiolipin antibody (Amer et al., Thrombosis Research 57 (1990)247-258) and a defective Factor VIII molecule (Dahlbäck et al., Thromb.Haemost. 65, Abstract 39, 658 (1991)).

WO 93/10261, in vitro methods for the diagnosis of a manifested bloodcoagulation disorder or for the screening of individuals predisposed toa blood coagulation disorder are disclosed. These methods are based onmeasurement of the anticoagulant response to exogenous APC added to aplasma sample from the individual to be tested, a weak anticoagulantresponse to APC, i.e., APC-resistance, indicating manifestation of orpredisposition to blood coagulation disorders, and especially athromboembolic disease. No explanation for APC-resistance is given butthe resistance to APC is suggested to be due to unknown interactions inthe blood coagulation system or to unknown coagulation factor(s)thereof. However, several possible explanations connecting theAPC-resistance to functional Protein S deficiency, a Protein Cinhibitory antibody, a protease inhibitor for APC or a mutation givingan APC-resistant Factor Va molecule or a Factor VIII gene mutation wereruled out.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates chromatography on Q-Sepharose (A) and Sephacryl S-300(B) of Factor V and APC-cofactor 2 activity.

FIG. 2 illustrates the results from characterization of isolatedAPC-cofactor 2 activity/Factor V on SDS-PAGE, Western blotting, andagarose gel electrophoresis.

FIG. 3 illustrates co-purification of APC-cofactor 2 activity and FactorV on monoclonal antibody affinity chromatography.

FIG. 4 illustrates correction of APC-resistance by purified APC-cofactor2 activity/Factor V.

FIG. 5 illustrates the elements and interactions of the human bloodcoagulation system.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention it has been found that APC-resistanceis due to deficiency of a previously unrecognized anticoagulant cofactoractivity enhancing the proteolytic effect of APC directed against FactorVa and Factor VIIIa. The findings that form the basis for the discoveryof the present anti-coagulant cofactor activity have been reported inDahlbäck et al., Proc. Natl. Acad. Sci. USA, 90 (1993) 1004-1008, saidreference having a publication date after the earliest priority dateclaimed for the present application.

More specifically, this anticoagulant activity has been found to beexpressed by Factor V, a finding that is quite surprising, since FactorV is the precursor to the procoagulant Factor Va, the latter beingdegraded by APC in the above mentioned Protein C anticoagulant system.Thus, Factor V is the second cofactor that has been found for APC, thefirst one being Protein S as mentioned above. Accordingly, the presentnovel anticoagulant cofactor activity is designated “APC-cofactor 2activity” or “Factor V anticoagulant activity” and, where appropriate,Factor V is also designated “APC-cofactor 2”. The prior known activityof Factor V is designated “Factor V procoagulant activity”. However, thepossibility that the said activity is associated with Factor Va cannotbe ruled out entirely.

The discovery of the novel anticoagulant cofactor activity according tothe present invention is based on the discovery of one patient withthrombosis and an abnormal APC-resistance when his plasma was assayedwith the methods disclosed in WO 93/10261 (incorporated herein byreference) and by Dahlbäck et al. (Thromb. Haemost. 65, Abstract 39(1991) 658). When studying a large cohort of patients with thrombosis,APC resistance was found to be the underlying cause in 30-40% ofidiopathic thromboembolic events (Thromb. Haemost. 69, 999, abstract(1993)).

Later, it has been found, according to the present invention, that acrude fraction obtained from normal plasma contained an activity, whichcorrected the defect of APC-resistant plasma, whereas the correspondingfraction from APC-resistant plasma from a patient with pronouncedAPC-resistance was inactive. This proves the existence of a novelcofactor to APC. In addition, by using preparations purified in thisactivity in assays, which have been designed to measure this activity,conclusive evidence for the existence of a novel cofactor to APC hasbeen achieved.

According to the present invention it has, thus, surprisingly been foundthat human Factor V has activity as a cofactor to APC, in addition toits well known function as a precursor to the procoagulant Factor Va.Possibly, this dual function of human Factor V is also expressed byFactor V derived from blood from some other animal species, especiallymammals, but not expressed in other species. For instance, all resultsso far obtained indicate that bovine plasma is lacking the saidactivity.

The said cofactor activity of Factor V means that Factor V enhances theproteolytic effect of activated Protein C, thus promoting thedegradation of Factor Va, i.e., the activated form of Factor V (FVa), aswell as the degradation of Factor VIIIa.

It is previously well known that the procoagulant activity of Factor Vis due to its activation by thrombin, in which three peptide bonds arecleaved, resulting in the formation of the procoagulant Factor Va as acomplex between the N- and C-terminal portions of the native Factor V.The function of the two large activation peptides derived from thecentral portion of Factor V is, however, unknown. As will be shown inthe experimental part of this disclosure, the APC-cofactor 2 activityhas not been found for Factor Va in the APC-resistance test used.

Thus, the APC-cofactor 2 activity is preferentially expressed by theintact Factor V molecule, probably the large fragments cleaved offduring activation thereof to Factor Va contributing to a major part ofsaid activity. However, the possibility that the activity is associatedwith a molecular entity which forms a highly stable complex with FactorV, and which is not split under the purification procedures used toisolate the Factor V having APC-cofactor 2 activity, cannot be ruled outentirely. Accordingly, in connection with the present invention, theexpressions “Factor V” and “Factor V having APC-cofactor 2 activity” andthe like are intended also to encompass a Factor V complex and alsofragments of Factor V, preferably other than the fragments originatingfrom thrombin cleavage of Factor V, having the said activity. ModifiedFactor V with retained APC-cofactor activity may also be obtainedthrough proteolytic cleavage by other enzymes of human or non-humanorigin such as snake venom enzymes and other proteases. Furthermore, theAPC-cofactor 2 activity was found to remain after partial proteolysis byan unknown enzyme during the purification thereof, indicating apotential existence of APC-cofactor 2 active Factor V fragments. Theexpressions APC-cofactor 2 as well as Factor V having anticoagulantactivity include fragments and subunits of Factor V/Va expressing theactivity or an immunologic determinant related to a region associatedwith the said activity. Although, for the sake of convenience,coagulation factors and the like are not described by species of originthroughout this description, such factors are preferably of human originunless otherwise specified.

In the experimental part of this disclosure, the procedures used forpurification and characterization of the present novel APC-cofactor 2activity are described, and its connection with Factor V is verified.

In summary, the evidence for the presence of the APC-cofactor 2 activityof Factor V is:

-   1. The procedure designed for the isolation of APC-cofactor 2    activity and earlier methods for the isolation of Factor V are very    similar. On SDS-PAGE, three bands appear at approximately 200-220    kDa (C-terminal portion), 140-160 kDa (N-terminal portion) and 330    kDa, which is very similar to what has been previously reported for    Factor V. (Compare the Examples below and Dahlbäck et al., J. Clin.    Invest. 66 (1980) 583-91.) The intensity of the band at 330 kDa is    enhanced for both APC-cofactor 2 activity and Factor V when higher    concentrations of protease inhibitors are used during the    purification procedure. For instance, a benzamidine hydrochloride    concentration of 10 mM gives rise to a significant band at 330 kDa.-   2. Specific polyclonal antiserum against human Factor V (Dakopatt    A/S, Denmark) reacts with each of the three bands associated with    APC-cofactor 2 activity in Western blotting.-   3. After addition of thrombin to the preparations comprising    APC-cofactor 2 activity, the three bands disappear and the products    obtained become indistinguishable from the products formed by    thrombin activation of Factor V.-   4. Seventeen monoclonal antibodies reacting with Factor V have been    obtained by using a preparation purified in respect of APC-cofactor    2 activity as immunogen. Two of the monoclonal antibodies partially    inhibited APC-cofactor 2 activity without inhibiting Factor V    procoagulant activity.-   5. Factor V procoagulant activity and APC-cofactor 2 activity are    coeluted on every chromatographic material tested: Heparin    Sepharose, Blue-Sepharose, Wheat Germ Lectin Sepharose, Q-Sepharose    and S-Sepharose (Pharmacia, Sweden).-   6. Both Factor V procoagulant activity and APC-cofactor 2 activity    are retained on a matrix carrying polyclonal antibodies against    human Factor V (Dakopatts A/S, Denmark).-   7. Both Factor V procoagulant activity and APC-cofactor 2 activity    are retained on matrices, such as Sepharose and Affigel, carrying    antisera against different fragments of bovine Factor V, which    cross-react with human Factor V.-   8. Both Factor V procoagulant activity and APC-cofactor 2 activity    are retained and coeluted on a chromatographic support, such as    Affigel, carrying a high affinity monoclonal antibody, which had    been prepared by using a preparation purified in respect of    APC-cofactor 2 activity as immunogen. In itself, this antibody    inhibited neither APC-cofactor 2 activity nor Factor V procoagulant    activity. Elution was performed at a pH of approximately 10.5-11.-   9. A recent publication disclosing that autoantibodies against    Factor V may result in thrombosis (Kapur et al., A. J. Hematol.    42 (1993) 384-388).

Preparations enriched in APC-cofactor 2 activity have been obtained bythe same methods as have been used previously for the isolation ofFactor V. It has been found that divalent metal ions, such as calciumions, have a stabilizing effect on the APC-cofactor 2 activity and,hence, calcium ions were added during the purification.

Essentially the same purification procedure was used in an attempt toelucidate the novel activity as was disclosed in the above mentioned WO93/10261. According to the results presented herein, the novel activityhas been identified as a cofactor activity to APC expressed as a novelproperty of Factor V, or, possibly, a complex or fragments thereof asdiscussed above. Thus, alternative and simpler preparation methods willbecome available. Current methods, such as gel chromatography, affinitychromatography with e.g., anti-APC-cofactor 2 activity antibody, asaffinity ligand, and ion exchange chromatography, etc., have been used,suitably after improvement to purify the novel activity describedherein. In addition, methods based on DNA-recombinant technique may beapplicable.

Accordingly, the present invention is also related to a preparationderived from blood or blood related products, such as plasma, saidpreparation being purified in respect of a blood coagulation component,which can express anticoagulant activity as a cofactor to APC therebyenhancing APC's proteolytic activity, directed against Factor V_(a) andFactor VIII_(a), said blood coagulation component being comprised ofFactor V or, optionally, a stable complex of Factor V and a molecularentity, which can express said activity.

The normal plasma level of Factor V is approximately 10-20 μg/ml. Byanalogy to other blood coagulation/anticoagulation factors, theAPC-cofactor 2 activity in 1 ml normal plasma is arbitrarily designated1 unit (U).

The present invention is also concerned with antibodies and antibodypreparations specific for a region of Factor V that is associated withAPC-cofactor 2 activity, i.e., a region in which there is a sitecarrying an epitope either causing APC-cofactor 2 activity orAPC-cofactor 2 inactivity. Such antibody preparations may be polyclonal,or preferably, monoclonal. Preferably, the antibodies of suchpreparations bind specifically to one or more Factor V sites associatedwith APCcofactor 2 activity. Alternatively, such a site could comprisean epitope involved in APC-cofactor 2 inactivity of Factor V and, thus,in APC-resistance. In connection with this invention the expression“epitope involved in APC-cofactor 2 inactivity” is meant to include anepitope related to decrease or loss of APCcofactor 2 activity.

Polyclonal antibodies can be obtained in accordance with known methodscomprising immunization of a suitable animal, such as a mouse, rat,rabbit, dog, horse, sheep, goat, bird (e.g., hen, chicken, etc.), with aproper immunogen and recovery of the present antibodies from anappropriate fluid derived from said animal, e.g., from blood or serum inthe case of mammals, or from eggs, when birds are immunized.

Preferably, the present antibodies are monoclonal antibodies which maybe obtained by conventional methods, e.g., essentially as disclosed byKöhler, G. and Milstein, C., Nature 256, 495 (1975). Generally, a methodto prepare monoclonal antibodies of the present invention includesimmunizing a mammal, preferably a mouse, with a proper immunogen,producing hybrid cells by fusion of lymphocytes, such as splenic cells,from the immunized mammal with myeloma cells, selecting fused cells in asuitable medium, screening antibody-producing cells, cloningantibody-producing cells, i.e., hybridoma, and producing monoclonalantibodies in ascitic fluid of mice or, optionally, in a culture mediumby propagation of the hybridoma therein. However, the present monoclonalantibodies, and fragments thereof binding to antigen, can also beobtained according to the methods based on recombinant technology, as iswell known in this art. In such methods, suitable host cells ofeucaryotic or procaryotic origin can be used. Such host cells are wellknown in this field of the art.

As immunogen, a purified preparation of Factor V, or fragments andderivatives thereof comprising the antigenic determinants responsiblefor expression of APC-cofactor 2 activity, can be used. Such fragmentsor derivatives may be conjugated to an immunogenic carrier, usually aprotein, to become antigenic.

Using human Factor V deficient in APC-cofactor 2 activity (which can beobtained as described below) as the immunogen combined with a two-stepscreening procedure for selecting hybridomas producing monoclonalantibodies reactive with the immunogen but not with normal intact humanFactor V, monoclonal antibodies reacting specifically with a humanAPC-cofactor 2 inactivity epitope, i.e., an epitope related to decreaseor loss of APC-cofactor 2 activity, may potentially be obtained.

A preferred embodiment of the present invention is related to monoclonalantibodies that bind to and also inhibit APC-cofactor 2 activity ofFactor V, at least in part. The present invention is also related toderivatives and fragments of such monoclonal antibodies, which are ableto bind to antigens.

According to the present invention, monoclonal antibodies produced bymouse/mouse hybridoma are preferred, since these are simple to obtain.Illustrative of such monoclonal antibodies are those antibodies producedby a novel hybrid cell line deposited on Dec. 8, 1993 in the PHLS Centrefor Applied Microbiology & Research, European Collection of Animal CellCulture, Salisbury, Great Britain with the provisional accession numberXAM-4-5-1 93120846. In connection with the present invention, monoclonalantibodies produced by this hybridoma are designated M4 (Master 4).

If not otherwise specified, the terms “antibody” or “antibodypreparation” encompass the intact antibody with its two heavy and twolight chains as well as different forms of derivatized antibodiescontaining the variable domains (F_(v)), e.g., fragments such as Fab,Fab′, F(ab′)₂; single chain antibodies; labelled antibodies, such asradiolabelled, fluorescent or enzyme-coupled antibodies; and antibodiesbound to solid phases, etc.

A further embodiment of the present invention is concerned with antibodypreparations, which comprise a definite number of monoclonal antibodiesof the above-mentioned specificity, such as 1, 2, 3, 4, 5 or moredifferent monoclonal antibodies. Such preparations may also bepolyclonal. Polyclonal and monoclonal antibody preparations directedspecifically against epitopes uniquely present in a site associated withAPC-cofactor 2 activity are potentially useful in immunoassays forspecifically determining the presence or absence of APC-cofactor 2activity in a sample (quantitatively and qualitatively).

The present invention is also related to hybridomas that produce themonoclonal antibodies of the present invention, and preferably to theabove mentioned hybridoma having the provisional accession numberXAM-4-5-1 93120846.

Although polyclonal and monoclonal antibodies specific for Factor V,which can be used to purify Factor V, are previously known, monoclonalantibodies deliberately raised against a region of Factor V associatedwith APC-cofactor 2 activity have not been disclosed before.

The antibody preparations (monoclonal as well as polyclonal) of thepresent invention may, in most cases, be used in purification proceduresbased on affinity chromatography in which antibodies of this inventionare attached to a solid carrier and used to selectively bind Factor Vpresent, e.g., in a plasma preparation. Subsequently, Factor V bound tothe solid carrier is eluted and collected.

The preferred monoclonal antibodies of this invention that bind toFactor V and inhibit, at least in part, APC-cofactor 2 activity ofFactor V, can be used to inhibit said activity of Factor V. Suchmonoclonal antibodies may, like the previously known anti-Factor Vantibodies, also be used to obtain plasma preparations deficient inAPC-cofactor 2 activity.

Important aspects of the present invention are concerned withtherapeutic methods, medicaments and pharmaceutical preparations, forwhich the knowledge of the novel anticoagulant activity of Factor V,i.e., APC-cofactor 2 activity, is used.

Accordingly, the present invention is also related to the use of FactorV, subunits or fragments thereof having anticoagulant activity ascofactor to APC for the manufacture of a medicament or pharmaceuticalpreparation intended for enhancing or restoring anticoagulant activityof APC in vivo. Specifically, such preparations are intended fortreatment of patients suffering from, or predisposed to, vasculardiseases, such as thromboembolic disorders including thrombosis anddisseminated intravascular coagulation (DIC).

Such a medicament or pharmaceutical preparation may be comprised of ahighly purified preparation of Factor V, which can be stored at lowtemperatures, such as −70° C.

The present preparations may also be used in connection with otherconditions or situations in which an individual would benefit from acorrected or enhanced blood anticoagulant activity, for instance, invarious clinical situations that are associated with increased risks forarterial and venous thrombosis.

Moreover, since the present APC-cofactor 2 activity is crucial for theeffect of APC, this activity may be used by itself or in combinationwith Protein C/APC and/or Protein S. Clinical situations where this mayprove to be important include patients being deficient in APC-cofactor 2activity, in particular in situations increasing the risks forthrombosis. In addition, supplemental APC-cofactor 2 activity may bebeneficial in connection with myocardial infarction, after thrombolytictherapy, in the post-operative period, in particular in high-riskpatients, as an adjuvant to patients treated for thrombosis, in patientsundergoing microsurgery, etc.

The administration route for APC-cofactor 2 activity is that normallyapplied for therapy with blood coagulation/anticoagulation factors, suchas intravenous or intra-arterial injection or infusion. As has beensuggested for other blood factors, oral administration can not beexcluded. The amount to be administered shall be effective in the sensethat, at least for a period of time, it fully or partially restores theeffect of the patient's own activated Protein C or the effect ofco-adminstered Protein C/activated Protein C, with the understandingthat even smaller effects may be beneficial to a patient at risk ofthrombosis. An amount in the range of 1-100, possibly 40-70, mg/day, canbe assumed to be useful. Repeated administration is preferred, becauseFactor V expressing APC-cofactor 2 activity is metabolized in themammalian body.

The different types of pharmaceutical compositions applicable are thesame as in use for other blood coagulation/anticoagulation factors, butadapted to the specific stability requirements that are necessary forFactor V having APC-cofactor 2 activity. For instance lyophilized orspray dried powders, optionally diluted with appropriate vehicles, aswell as sterile or aseptically produced aqueous solutions can be used.

A further aspect of the present invention is related to the use ofProtein C/activated Protein C and/or Protein S for the manufacture of apharmaceutical composition for the treatment of disorders related todeficiency in APC-cofactor 2 activity. The same types of compositions asintended for the prior art therapeutic use of Protein C and Protein Sare applicable.

Another aspect of the present invention is related to a Factor Vpreparation deficient in APC-cofactor 2 activity and is preferablyderived from humans. A potential therapeutic use thereof is in caseswhere an increase in Factor V_(a) activity over APC-cofactor 2 activityis beneficial to a patient.

The above-mentioned therapeutic methods and preparations are intendedfor mammals, particularly humans.

The novel anticoagulant cofactor activity according to the presentinvention can be used to develop methods for diagnosing such bloodcoagulation/anticoagulation disorders that are related to the functionalactivity of APC, and also to develop methods for monitoring and/ormeasuring functional activities of components involved in the bloodcoagulation/anticoagulation system, that directly or indirectly dependon the functional activity of APC.

Accordingly, a suitable embodiment of the present invention is relatedto a method for diagnosing a blood coagulation/anticoagulation disorder,preferably a thromboembolic disorder, or determining predispositiontherefor, in an individual, preferably a mammal, such as a human being,said method comprising determining in a sample, preferably a blood orblood derived sample, such as plasma, derived from said individual, thelevel of a blood component expressing anticoagulant activity, said bloodcomponent comprising Factor V, wherein the level of its anticoagulantactivity as a cofactor to APC is determined, an abnormal, preferably adecreased, level indicating manifestation of or predisposition to saiddisorder in particular for a decreased level said disorder being athromboembolic disorder.

Suitable embodiments of the above method are related to assaying theappropriate sample from an individual for Protein C/APC, Protein S orAPC cofactor 2 activity, and relating an observed abnormal level,preferably a lowered level, to a diagnosis that the individual has ablood coagulation disorder related to the assayed factor (i.e., toactivated protein C/Protein C, Protein S, or Factor V in its capacity asAPC-cofactor 2), which defect may be an underlying cause for athromboembolic disorder, or predispose to said disorder.

In the above methods, the level of the anticoagulant APC-cofactor 2activity is preferably measured in accordance with methods developedaccording to the present invention for assaying functional APC-cofactor2 activity that are described below. Immuno-based activity assays andnon-functional assays specific for Factor V carrying structural elementsassociated with its APC-cofactor 2 activity can also be used.

Thus, further aspects of the present invention are related to functionalassays for activated Protein C/Protein C, Protein S, and Factor Vexpressing APC-cofactor 2 activity and also to immune assays, nucleichybridization assays, and DNA and RNA sequencing methods for Factor Vexpressing APC-cofactor 2 activity.

These assays, as such, may have other uses than as diagnostics. Forinstance, the disclosed assays may be useful in monitoring purificationprocedures of components in the APC-cofactor system, and standardisingcontrol plasmas, etc.

A. Functional Assays of APC Protein C, APC-Cofactor 2 Activity andProtein S.

These assays utilize similar protocols as described earlier (Bertina etal., Res. Clin. Lab. 20 (1990) 127-138; Wolf et al., Thromb. Haemost. 62(1989) 1144-1145; WO 91/02812; WO 91/01382; WO 93/10261; Dahlbäck etal., Thromb. Haemost. 65, Abstract 39, (1991) 658).). The methoddisclosed in the US designation of WO 93/10261 (U.S. Ser. No. 199,328,issued on Aug. 25, 1995 as U.S. Pat. No. 5,443,960), which has beenincorporated by reference, comprises the following steps:

-   (i) a plasma sample obtained from the individual is incubated with    -   (a) an exogenous Reagent (I) activating at least partially the        blood coagulation system of the sample, and with    -   (b) activated exogenous Protein C (APC) or exogenous PC together        with exogenous Reagents (II) that transform PC to APC, and    -   (c) further components, such as Ca²⁺ salt and phospholipid or        tissue thromboplastin, that are necessary for efficient reaction        of the activated factors introduced according to step (i:a), and    -   (d) if desired, an exogenous substrate for an enzyme which        activity is influenced by activated Protein C;-   (ii) a substrate conversion rate is monitored directly for a blood    coagulation enzyme which activity is influenced by activated Protein    C,-   (iii) the conversion rate determined in step (ii) is compared with a    standard value being obtained from steps (i)-(ii) under identical    conditions for plasma of normal individuals.

As taught in U.S. Pat. No. 5,443,960, in cases where the substrateconversion rate is not normal compared to the standard, the individualfrom which the sample derives is classified as suffering from thedisorder or being at risk for acquiring the disorder. An increasedconversion rate of the sample indicates a thromboembolic disease or arisk for such a disease (with fibrinogen as the substrate an increasedconversion rate means a shortened clotting time). The significance of alowered conversion rate is at the present stage not known (withfibrinogen as the substrate a lowered conversion rate means a prolongedclotting time). Probably it is not related to any disease. The range ofthe normal conversion rate may be quite broad. Hence, it might, as acomplement, be of value to run steps (i)-(ii) on a plasma sample fromthe individual with exclusion of the incubation according to (i:b) andcompare the result obtained with that obtained.

In accordance with the present invention, a component in the system ofAPC, Protein S and Factor V, the latter in its capacity as APC-cofactor2, is assayed from the conversion of the appropriate APC substrate byAPC. Normal APC substrates are Factors V_(a) and/or VIII_(a), one orboth of which preferably are added to the assay medium as enriched, orhighly purified preparations, including preparations by recombinanttechnology, of unactivated (Factor V, Factor VIII) or activatedproteins. Within a series of samples that are to be compared, the assaymedia have essentially the same levels of:

(a) at least one of Factor V having APC-cofactor 2 activity or aninhibitor that blocks the same sample derived activity, and Protein S oran inhibitor that blocks sample derived Protein S activity, when APC orProtein C is to be assayed;

(b) at least one of Protein S or an inhibitor that blocks sample derivedProtein S activity, and APC, when APC-cofactor 2 activity is to beassayed; and

(c) at least one of Factor V providing APC-cofactor 2 activity or aninhibitor that blocks the same sample derived activity, and APC, whenProtein S is to be assayed.

Accordingly, the final assay media for a series of samples which are tobe compared contain sample and substrate for APC, and optionally one ortwo, preferably two, substances that do not derive from the sample andthat are selected from APC, Protein S or an inhibitor to Protein S, andFactor V having APC-cofactor 2 activity or an inhibitor to thisactivity, with the proviso that one of the remaining substances is theentity to be assayed (i.e., APC, Protein C, Protein S or APC-cofactor 2activity ).

The present method may comprise a) incubating in one or more steps in anaqueous assay medium, the sample and a substrate for APC, said substratebeing inherently present in the sample or added to the assay medium, andoptionally further blood coagulation components inherently present inthe sample or added to the assay medium, b) measuring the conversion ofthe substrate caused by APC during the incubation according to a), andc) correlating the measured value in a known manner to the activity tobe determined. In this method, optionally, one or two, preferably two,substances selected from APC, Protein S or a Protein S inhibitor, andFactor V having anticoagulant activity or an inhibitor to said activityare added to the assay medium of a), with the proviso that one of theremaining substances (i.e., APC, Protein S or APC-cofactor 2) is presentin the sample and is the component, the functional activity of which isto be determined (e.g., for Factor V, the activity being anticoagulantactivity as cofactor to APC).

Illustrative of other components that may be present are coagulationenzymes and other blood factors enabling the measurement of thedegradation of Factors V_(a) and/or VIII_(a). These other factors may beadded separately or may be present already in the sample. In case thesample contains Protein C, and APC is to be assayed, an activator forProtein C must be added. In case the sample contains varying levels ofcoagulation factors (other than the one to be assayed) interfering withthe assay reactions, one should secure excess of them (i.e., essentiallyconstant levels in the assay media) in order to avoid inter-samplevariations in the test results. For plasma samples, constant levels maybe accomplished by adding, in excess, normal plasma deficient in theentity to be assayed. The components to be added may also be in enrichedor highly purified forms. It can be envisaged that addition of FactorVIII/VIll_(a) and/or forms of Factor V not expressing the APC-cofactoractivity is suitable. Examples of forms that lack APC-cofactor activityare human Factor V deficient of the activity, Factor V from a speciesnot normally expressing the activity (for instance bovine Factor V, andFactor V fragments expressing Factor V activity but not APC-cofactor 2activity).

The addition of Protein S in the assay medium is done in order to avoidvariations in the measured level caused by intersample variations inProtein S, when APC-cofactor 2 activity or Protein C is to be measured.When Protein S is to be measured, APC-cofactor 2 activity may be addedfor the same purpose. The purpose of this is to keep the functionalactivity level of factors other than the one to be determinedessentially constant in the assay media on an inter-run basis. Aspreviously indicated, this may be accomplished by including into theassay media such factors in excess, for instance by adding normal plasmain excess, and/or by including functional excess of inhibitors for suchfactors, for example antibodies binding to epitopes responsible for theactivity of such factors. Thus, a monoclonal antibody specific to theepitopes responsible for the APC-cofactor activity of Protein S has beensuccessfully included (HPS 54, Dahlbäck et al., J. Biol. Chem. 265(1990) 8127-8235) in assay media for assaying APC-cofactor 2 activity.Similarly, functional inhibitors for APC-cofactor 2 activity, like theabove-mentioned monoclonal antibodies, may potentially be included inassay media when Protein S is to be assayed.

According to the present invention the functional assays are suitablyperformed in presence of added Factor VIII/VIII_(a).

The principles for the order of mixing, components to be added and thedifferent measuring principles are well-known in the field. See theabove-mentioned citations. Thus, APC activity may be followed byaddition of substrates such as fibrinogen (clotting assays) andchromogenic substrates for coagulation enzymes, the activity of whichare influenced by APC activity. Suitable chromogenic, fluorogenic andluminogenic substrates are thus thrombin and Factor X_(a) substrates.

The sample is normally plasma from an individual/patient, or the samplemay be Factor V having APC-cofactor 2 activity, Protein C (APC) orProtein S, all of these derived from a manufacturing process, orstandards to be used in the assay.

Native Factor V (abbreviated FV) produced through recombinant technology(rFV) may be used instead of FV purified from plasma as an adduct indiagnostic methods for Protein C/APC or Protein S, as a standard orcontrol in assays for FV anticoagulant activity, or as a therapeuticagent for administration to patients partially or completely deficientin APC-cofactor 2 activity. Alternatively, recombinant variants orfragments of FV with modified expressions of procoagulant oranticoagulant activity may be utilized for the same purposes and also asadducts in methods for FV anticoagulant activity. Such modifications maybe generated through mutations of the thrombin or APC cleavage sites inFV. In the former case the procoagulant activity, and in the latter casethe anticoagulant activity of FV, is partially or completely lost.Furthermore, such species, or suitable immunogenic peptide fragmentsthereof, may be used for preparation of monoclonal antibodies fordiagnostic or therapeutic use.

In assays for APC-cofactor 2 activity utilizing Factors V_(a) and/orVIII_(a) as the APC substrate and factors from the sample to measure APCsubstrate conversion, the sensitivity towards APC activity isconsiderably increased in plasmas from patients on treatment withvitamin K antagonists, resulting in an enhanced prolongation of clottingtime in certain clotting assays, especially APTT tests. The increasedsensitivity towards APC activity may be explained by the lowered levelsof vitamin K-dependent proteins such as Factors IX, X and II. SinceAPC-cofactor 2 activity is not vitamin K-dependent, it may thereforebecome possible to measure this activity in plasmas from such patientsby exogenous addition to the assay medium of certain vitamin K-dependentprotein(s), such as at least one of Factors IX, IX_(a), X and II,optionally combined with Protein S. These proteins may be added in theform of heavy metal salt eluate, such as a barium citrate eluate(Dahlbäck, Biochem. J. 209 (1983) 837-46) or aluminium hydroxide eluate(Bertina et al., Thromb. Haemost. 51 (1984) 1-5) or as purifiedcomponents before measuring the APC substrate conversion. If the plasmacontains heparin (standard or of low molecular weight) it is suitable toneutralize this effect by adding excess of heparin, or by addingpolybrene or Protamine, or the like, as heparin inhibitors to reduceinterference with the assay results.

As stated above, the present methods for determining functionalactivities of PC/APC, Protein S, or Factor V anticoagulant activity aresimilar to methods described earlier, e.g., in the cited references, thedisclosure of which is included herein by reference. Thus, a detaileddescription of these methods should not be required. In principle,however, such methods are based on measurement of conversion of asubstrate, the rate of which can be directly or indirectly determinedand related to the substance to be assayed, e.g., based on coagulationor chromogenic assays, suitably in presence of further componentsnecessary to detect the conversion rate, which are inherently presentin, or added to, the sample.

Such components may comprise a reagent that serves to introduce anactivated coagulation factor that can be used for determination of thesubstrate conversion rate. This reagent leads to the presence of atleast Factor IX_(a), and may comprise a certain coagulation factor or areagent that activates the system via the intrinsic or extrinsicpathway. Accordingly, this reagent may be Factor IX_(a) or FactorXI_(a), (intrinsic pathway), Factor XII_(a) (intrinsic pathway),kallikrein (intrinsic pathway), a contact activator (intrinsic pathway)such as kaolin, celite or ellagic acid (intrinsic pathway), an APTTreagent (Activated Partial Thromboplastin Time; i.e., a reagentcontaining a phospholipid and a contact activator (intrinsic pathway)),tissue thromboplastin (PT-reagent, PT=Prothrombin time (extrinsicpathway)), tissue factor, Factor VII_(a) and Factor X_(a).

Other components that can be added, depend on the mode employed and maynecessitate the inclusion of plasma protease inhibitors for enzymesother than the monitored one or the inclusion of a fibrin polymerizationinhibitor. Ca²⁺ may be in the form of a plasma soluble salt thatprovides the Ca²⁺ ion in free uncomplexed form, i.e., strong Ca²⁺ ion infree uncomplexed form. Such additional components suitably also includeFactor VIII/VIII_(a) and Factor V/V_(a).

The substrate for which the conversion rate is determined may comprise asynthetic substrate for an enzyme, the activity of which is influencedby activated Protein C, for example thrombin (Factor II_(a)) and FactorX_(a). Suitable synthetic substrates are water soluble and preferablyhave an oligopeptide structure with three, four or five amino acidresidues and an amino terminus that is protected from being attacked byamino peptidases. The protection is accomplished either by a protectinggroup or by having a D-amino acid at the amino terminus. In order togive a detectable response, the carboxy terminus of a syntheticsubstrate is amidated with a group that specifically can be released anddetected upon action of the relevant blood coagulation protease. Thegroup to be released is selected among chromogenic, fluorogenic orchemiluminogenic groups and other analytically detectable groups. Seefurther H. C. Hemker, “Handbook of Synthetic Substrates for theCoagulation and Fibrinolytic System”, Martinus Nijhoff Publishers, 1983,and J. Fareed et al., “Synthetic Peptide Substrates in HemostaticTesting” in CRC Critical Reviews in Clinical Laboratory Sciencies Vol19, Issue 2, 71-134 (1983). In the case of samples other than plasmasamples, exogenous fibrinogen may be added as substrate.

In order to accomplish a specific result with respect to the substanceto be determined, in some cases one should try to keep the plasma samplecontent of the final assay medium as high as possible. Accordingly, aplasma sample content in tests having good specificity could be >10%, inparticular >20% or >35% (v/v). In other cases, however, an essentiallylower content, that is, below 10% (v/v), can be used.

B. Immune Assays for APC-Cofactor 2 Activity.

The antibody preparation of the invention will enable immune assays ofAPC-cofactor 2 activity, wherein anti-APC-cofactor 2 antibody is allowedto form an immune complex with Factor V having APC-cofactor 2 activityin the sample in an amount that is a qualitative or quantitative measureof the APC-cofactor 2 activity level in the sample. The samples may bethe same as for functional assays. The present invention is alsoconcerned with reagents for use in assays described herein.

Purified preparations comprising Factor V expressing the APC-cofactor 2activity, which has been purified from plasma or prepared by recombinanttechnology, Protein C preparations, optionally in an activated form orcombined with a Protein C activator, and Protein S preparations, whichcontain defined amounts of their respective factor may be used as areagent, a standard or a control in the above-mentioned assays. TheProtein C preparation may be combined with at least one vitamin Kdependent coagulation factor selected from Factors IX, X and II, andoptionally combined with Protein S. Products and preparations fortherapeutic use may also be obtained by recombinant technology.Furthermore, the present monoclonal antibodies may be obtained byrecombinant technology, and essentially PCR-technology, which is wellknown, may be used to obtain such antibodies having desired specifity.

There are indications that information may be obtained about variousFactor V mutations based on interactions between Factor V anticoagulantactivity and Protein S. Methods may be designed to obtain suchinformation in the presence or absence of a suitable antibody. Suchmethods in the presence of antibody may be used as a quantitative methodfor an analyte, such as Factor V anticoagulation activity and Protein S.

C. Hybridization Assays.

Recent results have shown in a conventional DNA-linkage study of a largefamily with inherited APC resistance that there is a strong linkagebetween a neutral polymorphism in the Factor V gene and expression ofAPC-resistance. This strongly suggests that a mutation in the Factor Vgene is the cause for APC-resistance. This is conclusive evidence thatnucleic acid hybridisation assays, as well as nucleic acid sequencingcan be used in conventional ways in order to detect individuals at riskfor thrombotic events due to a low level of APC-cofactor 2 activity.Thus, these types of assays may be used for checking, in an individual,the abnormal presence or absence of one or more nucleic acid fragment(s)and/or sequence(s) unique for the presence or absence of expression of aFactor V molecule either carrying APC-cofactor 2 activity or beingdeficient in this activity. The protocols and conditions are the same asnormally applied for other genes, except for now using reagents specificfor the Factor V gene and, optionally, mutation(s) associated withAPC-resistance or specific for a normal Factor V gene. Any cell samplefrom the individual may be appropriate.

Furthermore, the present invention is concerned with Factor V, suitablyhuman Factor V, capable of becoming activated to exert Factor V_(a)procoagulant activity but not capable of exerting anticoagulantactivity, preferentially not anticoagulant activity as a cofactor toAPC, said factor being in a substantially pure form.

Another aspect of the invention is related to Factor V, suitably humanFactor V, capable of exerting anticoagulant activity, preferentially asa cofactor to APC, but not capable of expressing procoagulant activityof Factor V_(a).

Such Factors can be purified from plasma with methods similar to normalFactor V, or prepared by recombinant technology. Possible applicationsare in standards and as supplementing reagents, and for therapeutic use.

EXAMPLES

Assay for APC-Cofactor 2 Activity:

A modification of the recently described APC-APTT method(PCT/SE/9200781; and Dahlbäck et al., Proc. Natl. Acad. Sci. USA, 90(1993) 1004-1008) was developed to measure APC-cofactor 2 activityduring its purification. The method used plasma from an individual whichhad an inherited poor response to APC and fractions obtained from normalplasma which were tested for their ability to normalize the poor APCresponse. The assay which will be referred to as APC-cofactor 2 activityassay was performed as follows: 50 μl plasma demonstrating a poorresponse to APC (referred to as APC-resistant plasma) was incubated with50 μl of the test fraction and 50 μl of an activated thromboplastin time(APTT) reagent (APTT-automated Organon Technica (USA)) for 5 minutes at37° C. before coagulation was initiated by the addition of 5 μl of anAPC-CaCl₂ mixture (if not indicated otherwise, 20 nM human APC in 10 mMTris-HCI, 0.05 M NaCl, 30 mM CaCl₂, pH 7.5, containing 0.1% bovine serumalbumin (BSA)), and the coagulation time was measured. The presence ofAPC-cofactor 2 activity in a test sample is associated with an increasein clotting time.

Each example was also analyzed in parallel without the addition of APCto the CaCl₂ solution and the APC-dependent prolongation of clottingtime was calculated. To construct a dose-response curve for APC-cofactor2 activity, the plasma deficient in APC-cofactor 2 activity was mixedwith control plasma and used as test-plasma in the APC-APTT method. Theanti-coagulant response of APC was related to the percentage of controlplasma and the curve had an exponential shape. As it was unknown whetherthe plasma deficient in APC-cofactor 2 activity was completely devoid ofFactor V expressing APC-cofactor 2 activity, the assay only provided asemi-quantitation of the cofactor in different fractions. However, theassay served the purpose of providing a means to follow the APC-cofactor2 activity during its purification.

A Factor V clotting assay was performed using Factor V-deficient plasmaas described previously (J. Clin. Invest. 66, 583-591 (1980)). Thepresence of Factor V activity resulted in a shortening of clotting timeof the deficient plasma. In both the APC-cofactor 2 activity assay andFactor V clotting assay the original clotting data have been shownrather than the results converted into units.

Electrophoretic Immunological and Other Methods: Gradient (5-15%)polyacrylamide slab gel electrophoresis in the presence of sodiumdodecyl sulfate (SDS-PAGE) and Western blotting were performed usingtechniques previously described (J. Biol. Chem. 261, 9495-9501 (1986)).A specific rabbit polyclonal antiserum against Factor V was the kindgift of Dakopatts A/S. Data demonstrating the specificity of theantiserum have been reported previously (Blood 68, 244-249 (1986)).Rabbit polyclonal antibodies were raised against the isolated heavy andlight chain fragments of bovine Factor V (J. Biol. Chem. 261, 9495-9501(1986)). Monoclonal antibodies were raised using standard methods, aspreviously described in detail (J. Biol. Chem. 265, 8127-8135 (1990)).The purified protein in the S-300 pool was used as antigen in theimmunization of Balb/c mice. Seventeen different antibodies wereobtained and their reactivities tested with Western blotting.Approximately 20 mg of an antibody designated Master 30 was coupled to 4ml Affigel 10 (Biorad) in accordance with the manufacturer'sinstructions. IgG-fractions of the polyclonal antisera against humanFactor V and the bovine Factor V fragments were also coupled to Affigel(approximately 5 mg/ml).

Purification of APC-Cofactor 2 Activity:

All manipulations of samples were performed on an ice bath;chromatographies and centrifugations were run in the cold room, suitablyat 4° C. Blood-collection: Human freshly frozen (−70° C.) citratedplasma was obtained from the local blood bank. The frozen plasma (2.3 L)was thawed at 37° C. and the following protease inhibitors were added:phenylmethanesulfonyl fluoride (PMSF) (1 mM), diisopropylfluorophosphate(DFP) (1 mM), soy bean trypsin inhibitor (50 mg/L), Trasylol (aprotinin)(1.5 mg/L which is equal to 10 units/ml), and benzamidine (10 mM). Theplasma (kept on an ice-bath) was subjected to barium-citrate adsorptionas previously described (Dahlbäck, Biochem. J. 209 (1983) 837-846) andthe barium-adsorbed plasma was subjected to fractionated polyethyleneglycol precipitation (PEG 6000) (8% w/v) by the addition of solid PEG.The APC-cofactor activity was recovered in the 8% PEG supernatant. The8% PEG supernatant was diluted with an equal volume of 10 mM benzamidineand then mixed with Q-Sepharose (Pharmacia LKB Biotechnology, Uppsala,Sweden) and equilibrated in 20 mM Tris-HCl, 0.1 M NaCl, 1 mM CaCl₂, pH7.5, comprising 10 mM benzamidine. After 1 hr of gentle mixing, the gelwas collected in a Buichner funnel and washed with A, 3 L equilibrationbuffer, B, 1 L equilibration buffer with 0.1% Tween 20 and C, 2 Lequilibration buffer containing 0.15 M NaCl instead of 0.1 M NaCl. Thegel was then packed in a column (5 cm diameter) and the adsorbedproteins were eluted with a linear gradient of NaCl (0.15-0.5 M NaCl in20 mM Tris-HCl, 1 mM CaCl₂, 10 mM benzamidine, pH 7.5, 1.5 L in eachgradient vessel). The flow rate was 330 ml/h and 11 ml fractions werecollected. Fractions were analyzed for APC-cofactor 2 activity andFactor V activity in 1/10 dilutions (FIG. 1).

Fractions were pooled as indicated by the horizontal bar and subjectedto (NH₄)₂SO₄ precipitation (70% saturation). The precipitate wascollected by centrifugation, dissolved in a minimal volume of 20 mMTris-HCl, 0.15 M NaCl, 1 mM CaCl₂, pH 7.5, containing 10 mM benzamidine,1 mM DFP, and 1 mM PMSF and applied to a column (2.5 cm×93 cm) withSepharcryl S-300 (Pharmacia, Uppsala, Sweden) equilibrated in the samebuffer but without DFP and PMSF. The column was run at 10 ml/h and 1.2ml fractions were collected. The fractions were analyzed withAPC-cofactor 2 activity assay and Factor V assay at 1/10 dilutions (FIG.1). Fractions were pooled as indicated by the horizontal bar and storedat −70° C.

Affinity Chromatography Using Monoclonal Antibodies

The protein obtained as described above from an S-300 chromatography (inthe illustrated run approximately 6 mg in 20 mM Tris-HCl, 0.1 M NaCl, 2mM CaCl₂, pH 7.5) was applied to a column (0.75 cm×7.5 cm) of Affigelwith immobilized monoclonal antibody designated Master 30, the columnand protein being equilibrated in 20 mM Tris-HCl, 0.1 M NaCl, 2 mMCaCl₂, pH 7.5. After washing the column until absorbance of the eluatereached zero, bound proteins were eluted with 50 mM diethanolamine, 2 mMCaCl₂, pH 10.6. The pH of the eluate was immediately neutralized with 3M Tris-HCl, ph 7.5 (50 μl per 1 ml fraction). The fractions wereanalyzed (at ⅕ dilution) with APC-cofactor 2 activity assay and Factor Vclotting assay. Active fractions were pooled, concentrated byultrafiltration (YM10 membranes) and stored at −70° C. The purifiedAPC-cofactor 2/Factor V was activated with thrombin as describedpreviously (J. Clin. Invest. 66, 583-591 (1980)).

Preparation of Monoclonal Antibodies

The purified protein, i.e., Factor V (APC-cofactor 2), was used as animmunogen for the immunization of Balb/c mice in accordance with astandard protocol. Splenic cells from said mouse were fused with cellsof the Sp 2/0 Ag14 mouse myeloma cell line and selected inhypoxanthine-aminopterin-thymidin DMEM medium as disclosed by Köhler andMilstein (supra).

A solid phase enzyme-linked immunosorbent assay (ELISA) was used todetect antibodies produced against Factor V in antisera from the mice aswell as to detect antibody-producing hybrid cells. In those assays,Factor V (10 μg/ml in standard coating buffer) was coated in wells onmicrotiter plates. Antisera from immunized mice and supernatants of thehybrid cell cultures were added in dilution to the wells and individualwells were assayed for the presence of antibodies bound to Factor V withthe aid of an enzyme-labelled secondary antibody by standard methods.

Hybrid cells from positive wells, i.e., antibody-producing cells, werecloned by limiting dilution, subcloned and expanded. After implantationin the abdominal cavity of pristane pretreated mice, monoclonalantibodies were produced in ascitic fluid in large amounts.

Seventeen masters were obtained, all of which reacted with antigenicdeterminants on Factor V as shown in accordance with the Western blotmethod, the majority of these monoclonal antibodies (abbreviated Mab's)being directed to the same region of Factor V, namely, the activationfragment comprising the central 150 kDa region of Factor V.

One of these Mab's, designated Master 30, was used for the affinitypurification of Factor V as described above. The Mab's described abovewere tested to determine their influence on coagulation activity andAPC-cofactor 2 activity in plasma.

Increasing amounts of purified Mab up to 400 μg/ml were added to normalplasma and after incubation (15-30 minutes), the activity of Factor Vwas measured with a conventional Factor V assay based on coagulationanalysis, and the response to exogenous APC was determined according tothe following.

Normal plasma samples comprising varying concentrations of Mab (10-400μg/mL) were incubated with a commercial APTT reagent. (In the presenttests Automated APTT from Organon was used. Similar results wereobtained with the APTT reagent from COATEST APC Resistance, ChromogenixAB, Mölndal, Sweden.) After incubation for 5 minutes at 37° C. either 30mM CaCl₂ (in 20 mM Tris-HCl, 50 mM NaCl, pH 7.5 comprising 0.1% bovineserum albumin (BSA)) or activated Protein C (APC) (about 2 μg/ml in 30mM CaCl₂ dissolved in 20 mM Tris-HCl, 50 mM NaCl, pH 7.5 comprising 0.1%BSA) was added and the clotting times were recorded. The APTT assay wasperformed essentially as disclosed by Dahlbäck et al., PNAS 90 (1993)1004-1008.

The presence of these Mab's either had no effect on the conventionalAPTT time, i.e., the clotting time, obtained for samples comprisingadded CaCl₂, or had only a moderate effect, with clotting times of 40-45seconds being observed. Two of the Mab's, designated Master 1 and Master4, were, however, found to shorten the clotting time for samples, towhich APC in a CaCl₂ solution had been added, (APC time).

The following clotting times were obtained:

APC time in the absence of Mab's 110-120 seconds

APC time in the presence of Master 4 80-90 seconds.

These results indicate an inhibition in part of the APC-cofactor 2activity in plasma in the presence of Master 4. This partial inhibitionactivity of Master 4 was found to be dependent on the added amount,maximal inhibition being obtained when 50-100 μg of Master 4 per mlplasma were added. Master 4 has been deposited as stated above.

The results from the above tests are discussed below with reference tothe Figures.

FIG. 1 illustrates chromatography on Q-Sepharose (A) and Sephacryl S-300(B) of factor V and APC-cofactor 2 activity. On both columns, theelution profile of APC-cofactor 2 activity (upper sections) coincidedwith that of Factor V (middle sections). Factor V activity wasdemonstrated as a shortening of clotting time of Factor V-deficientplasma, whereas APC-cofactor activity was associated with anAPC-dependent prolongation of clotting time of APC-resistant plasma. Thefractions were pooled as shown by the horizontal bars.

FIG. 2 illustrates the results from characterization of isolatedAPC-cofactor 2/Factor V on SDS-PAGE, Western blotting, and agarose gelelectrophoresis. The pool from the S-300 column was analyzed bySDS-PAGE, before and after incubation with thrombin. The gels wereeither stained with Coomassie blue (A) or subjected to Western blottingusing monoclonal antibody (Master 30) (B) or polyclonal (C) antibodies.Samples applied to the SDS-PAGE were reduced; approximately 20 μgprotein was applied to each lane in the protein-stained gel, whereasapproximately 1 μg was applied to each of the lanes used for Westernblotting. Lanes with thrombin-cleaved protein are marked T. Positions ofmolecular weight markers are given to the left. Factor V-relatedpolypeptides are marked with arrows, whereas fragments formed bythrombin (J. Biol. Chem. 257, 6556-6564) are indicated by arrowheads.The 150 kDa fragment stains poorly with Coomassie, but is readilyobserved on Western blotting. Intermittently observed bands are denotedby asterisks. The S-300 pool was also analyzed by agarose gelelectrophoresis (bottom section). The positions of albumin (alb), α₁,α₂, β₁ and β₂ bands of a plasma control are indicated by vertical lines.

FIG. 3 illustrates copurification of APC-cofactor 2 activity and FactorV on monoclonal antibody affinity chromatography. The S-300 pool wasapplied to monoclonal antibody (Master 30) affinity chromatography. Asthe binding capacity of the column was exceeded, most of the proteinpassed through the column. After washing the column, the bound proteinwas eluted with high pH (start of elution indicated by arrow). Fractionswere analyzed with both APC-cofactor 2 activity and Factor V assay.Factor V activity was associated with a shortening of clotting time ofFactor V-deficient plasma, whereas APC-cofactor activity gave anAPC-dependent prolongation of clotting time of APC-resistant plasma. Thetwo dashed lines represent clotting times of buffer controls.

FIG. 4. A-B illustrates correction of APC-resistance by purifiedAPC-cofactor 2/Factor V. Affinity purified APC-cofactor 2/Factor V (atindicated concentrations in a volume of 50 ul) was mixed withAPC-resistant plasma (50 ml). The mixtures were then tested in theAPC-cofactor 2 activity assay (A) with (●) and without (∘) APC in theCaCl₂-solution, and in the Factor V assay (B). Each point represents themean of duplicate measurements.

RESULTS

APC-cofactor 2 activity was analyzed with a biological assay usingplasma from an individual (designated AS-plasma) with APC-resistance astest plasma, and a procedure was devised for purification ofAPC-cofactor 2 from normal plasma. The first step in the procedure wasbarium-citrate adsorption, which removed the vitamin K-dependentproteins including Protein C and Protein S. The barium-citrate eluatehad no APC-cofactor 2 activity. On fractionation of the supernatantplasma with PEG 6000 precipitation, the APC-cofactor 2 activity waspresent in the 8% PEG supernatant, whereas the dissolved 0-8% PEG 6000precipitate had no APC-cofactor 2 activity. The APC-cofactor 2 activityin the 8% PEG supernatant was purified first by anion exchangechromatography on a column with Q-Sepharose and then by gel filtrationon Sephacryl S-300 (FIG. 1). This purification protocol was very similarto a procedure for purification of coagulation Factor V (J. Clin.Invest. 66 583-591 (1980)), and Factor V was found in the same fractionsof APC-cofactor 2 activity. The purification was performed at least 10times with different modifications, and the elution profiles for FactorV and APC-cofactor 2 activity were consistently very similar. Theprotein in the S-300 pool expressed both Factor V and APC-cofactor 2activities, and manifested characteristics previously reported forFactor V (J. Clin. Invest. 66 583-591(1980)). Additional efforts toseparate the two activities using several other chromatographicprinciples, such as Heparin Sepharose, Blue Sepharose and Wheat germagglutinin Sepharose were unsuccessful (not shown), and APC-cofactor 2activity was in fact found to purify together with Factor V on everychromatographic support that was tried.

SDS-polyacrylamide gel electrophoresis of the protein in the S-300 poolyielded a 330 kDA band (corresponding to single chain Factor V) inaddition to bands with molecular weights of approximately 220,000 and130-150,000 (FIG. 2). These bands represented cleaved Factor V and, likethe 330 kDa species, they reacted with a polyclonal antiserum againstFactor V on Western blotting (FIG. 2). The 220 kDa band represented theC-terminal part of Factor V, including the 74 kDa light chain of FactorV_(a) and the larger (150 kDa) of the two activation fragments, and wasrecognized by an antiserum against the light chain of bovine FactorV_(a) (results not shown). The 130-150 kDa bands comprised theN-terminal part of Factor V (105 kDa heavy chain plus the smaller of thetwo activation fragments), and accordingly reacted with an antiserumagainst the bovine Factor V_(a) heavy chain (results not shown).Additional bands of lower molecular weights, which did not react withpolyclonal Factor V antiserum on Western blotting were sometimes seen,but when present, their elution profiles (as judged by SDS-PAGE) on theS-300 chromatography did not correlate with the activity of Factor V orwith APC-cofactor 2 activity. Incubation of the purified protein withthrombin yielded fragments characteristic for thrombin-cleaved Factor V,and concomitantly the activity in the APC-cofactor 2 assay was lost,suggesting APC-cofactor 2 activity only to be expressed by Factor V andnot by Factor V_(a). On Agarose gel electrophoresis, the purifiedprotein migrated as a single species to an inter-alpha position (FIG.2), and both Factor V and APC-cofactor 2 activities could be eluted fromthis position of the gel (not shown).

As Factor V is extremely sensitive to proteolysis, an abundance ofprotease inhibitors was included in the final protocol. When performedin the absence of protease inhibitors, the purification procedureresulted in a more degraded product lacking the 330 kDa species, butcontaining the 220 kDa and 130-150 kDa bands. This purified productexpressed both Factor V and APC-cofactor 2 activities. Factor V requirescalcium for its stability; and when calcium was not included in thepurification, both Factor V and APC-cofactor 2 activities were graduallylost.

The protein in the S-300 pool was used as antigen, as described above,for the production of monoclonal antibodies. Seventeen antibodies wereobtained, and they were all found to react with the 330 kDa single chainFactor V as well as with the 220 kDa species, as judged by Westernblotting (FIG. 2). After thrombin cleavage of Factor V, all antibodiesreacted with the 150 kDa activation fragment (the larger of the twoactivation fragments).

One of the antibodies (Master 30) was immobilized on Affigel and usedfor affinity chromatography (FIG. 3). The S-300 pool was applied to thecolumn. The protein that bound to the column was eluted and found tohave both Factor V and APC-cofactor 2 activities. The elution profilesof both activities coincided, but manifested considerable trailing.Other elution conditions such as using higher or lower pH, or denaturingagents were tried but were unsuccessful as they resulted in loss of bothactivities. The S-300 pool was also applied to columns with immobilizedpolyclonal antibodies against human Factor V or against bovine FactorV_(a) fragments. Both Factor V and APC-cofactor 2 activities wereretained on the columns, but the denaturing conditions required to elutethe bound protein resulted in loss of both biological activities(results not shown).

Increasing concentrations of affinity purified APC-cofactor 2/Factor Vwere added to AS plasma and the anticoagulant response to APC tested. Adose-dependent increase in anticoagulant response to APC was observed(FIG. 4A). Approximately 25 mg/L, which is of the same order ofmagnitude as the normal plasma concentration of Factor V, was requiredto yield an APC-response of AS plasma comparable to that of normalplasma (clotting times in the presence of APC of 90-110 seconds). Theaffinity purified protein was also active in Factor V assay, asdemonstrated by a shortening of the clotting time (FIG. 4B).

Assays for Components in the APC-Cofactor System

The following examples show that by keeping the levels constant of twoof the components in the APC-cofactor system comprised of APC, Factor Vhaving APC-cofactor 2 activity and Protein S, and varying the remainingone, different substrate conversion rates will be achieved. This impliesthat assays as outlined above for each of the components can beconstructed. An assay employing plasma deficient in APC-cofactor 2activity has been disclosed above.

Effect of APC-Cofactor 2 in a Chromogenic Assay.

The assay principle is based upon the monitoring of the degradation ofFVIII_(a) by APC through the FIX_(a)-dependent activation of FX, inwhich system FVIII_(a) serves as an important cofactor to FIX_(a). Thus,a decreased level of FVIII_(a) will result in a decreased generation ofFX_(a), determined through the hydrolysis of a FX_(a)-sensitivechromogenic peptide substrate.

-   I. 50 μL of a normal plasma dilution 1:20 in 50 mmol/L Tris-HCl    buffer, pH 7.3, I=0.15 and 1% bovine serum albumin (BSA) containing    highly purified FVIII concentrate (Octonativ M®, Kabi Pharmacia AB,    Stockholm Sweden), 2 IU/mL, was mixed with 50 μL bovine thrombin,    0.06 nkat/mL (activity vs. the substrate S-2238, (Chromogenix AB,    Mölndal, Sweden)) for 30 s at 37° C.-   II. Thereafter 100 μL of a reagent (R) mixture containing 40 mmol/L    Tris-HCl, pH 7.3 and 0.15% BSA, CaCl₂, 12 mmol/L, and phospholipids,    30 μmol/L, as well as other components defined below, was added to    the above mixture, followed by an incubation for 2 min at 37° C.-   III 25 μL was then subsampled from this mixture and diluted with    1000 μL 50 mmol/L Tris-HCl buffer, pH 7.3, I=0.15 with 0.2% BSA,    followed by analysis of FVIII activity according to the COATEST®    FVIII assay principle (Chromogenix AB, Mölndal, Sweden).-   IV. 200 μL of a reagent containing bovine FIX_(a) and bovine FX    (COATEST FVIII, Chromogenix AB, Mölndal, Sweden) and phospholipids,    30 μmol/L, was mixed with 100 μL of the diluted subsample and with    100 μL CaCl₂, 25 mmol/L. After 5 minutes incubation at 37° C., 200    μL of the chromogenic FX_(a) substrate S-2765 (Chromogenix AB,    Mölndal, Sweden), 0.9 mmol/L was added. After further 3 min    incubation at 37° C., the substrate hydrolysis was stopped by    addition of 100 μL acetic acid, 20%, and the absorbance of the    released Chromophore pNA (p-nitroaniline) was read at 405 nm in a    photometer.

In this assay system, the concentration of active FVIII in the sample isdirectly proportional to the absorbance. The content of supplementarycomponents in the different R-mixtures are:

A. None

B. APC, 0.4 μg/mL

C. APC, 0.4 μg/mL+APC-cofactor 2 activity, 0.3 U/mL

D. APC, 0.4 μg/mL+human Protein S, 1 μg/mL

E. APC, 0.4 μg/mL+human Protein S, 1 μg/mL+APC-cofactor 2 activity, 0.3U/mL

Normal plasma contains approximately 10 μg/mL of free Protein S, hencethe sample dilutions contributes with 0.05×0.05×10=0.025 μg in stage II,corresponding to one fourth of the added amount of purified humanProtein S. The content of APC-cofactor 2 activity should be consideredas an approximate estimation since no quantitative method yet exists.Effect of APC-Cofactor 2 activity Protein S, conc. on APC activityR-Mixture in stage II, μg/mL A 405 expressed as Δ405 A 0.125 0.678 B0.125 0.623 C 0.125 0.509 −0.114 (C − B) D 0.625 0.559 E 0.625 0.389−0.170 (E − D)

Thus, the results show that addition of APC-cofactor 2 activity enhancesthe activity of APC at both levels of Protein S, illustrated as adecrease in the FX_(a)-generation, i.e., an increased rate ofinactivation of FVIII_(a) in stage II.

Effect of APC-Cofactor 2 Activity in Clotting Assay.

Cofactors FV_(a) and FVIII_(a) are involved in the generation ofthrombin, the enzyme responsible for fibrin formation. These cofactorsare degraded by APC and hence the activity of APC is illustrated in aclotting assay as a prolongation of the time needed for generation ofthe fibrin clot. Since Protein C (PC) circulates as a proenzyme,activation of PC in the sample is accomplished by addition of the snakevenom enzyme Protac C® (Pentapharm, Basel, Switzerland). The followingexperiment was performed:

-   I. 10 μL FVIII concentrate (Octonativ M®, Kabi Pharmacia AB,    Stockholm, Sweden), 10 IU/mL, was mixed with 100 μL PC-deficient    plasma, 100 μL APTT reagent, 25 μL Protac C®, 1.5 U/mL, and 25 μL of    a reagent (R) mixture containing 50 mmol/L Tris-HCl, pH 7.5, I=0.15,    0.2% BSA and further components defined below, was added to the    above mixture. The complete mixture was incubated for 4 min at 37°    C.-   II. 100 μL CaCl₂, 22 mmol/L, was then added to the above mixture and    the time needed for clot formation at 37° C. was recorded.-   Supplementation in R-mixtures:    -   A. None    -   B. PC, 2 μg/mL    -   C. PC, 2 μg/mL, +APC-cofactor 2 activity, 2.6 U/mL    -   D. PC, 4 μg/mL    -   E. PC, 4 μg/mL, +APC-cofactor 2 activity, 2.6 U/mL    -   F. APC-cofactor 2, 2.6 U/mL

The Protein C deficient plasma contributes the other plasma proteinsinvolved in the clotting process as well as Protein S, a cofactor forAPC. The final concentration of APC-cofactor 2 activity in stage I isapproximately 0.2-0.3 U/mL (see above). Concentration Prolongation ofAPC of PC in activity due to R-Mixture stage I, μg/mL Clotting time, sAPC-Cofactor 2, s Δ A 0 42.3 ± 0.7 (n = 5) B 0.2 62.7 ± 1.2 (n = 5) C0.2 71.4 ± 1.6 (n = 3) 8.7 D 0.4 79.3 ± 2.9 (n = 5) E 0.4 104.5 ± 8.6 (n= 3)  25.2 F 0 45.9

Thus, the experiments clearly show that addition of APC-cofactor 2activity enhances the APC activity, expressed as an increasedprolongation of the clotting time. The effect of the addition of theAPC-cofactor 2 preparation in the absence of PC is only minor.

1-43. (canceled)
 44. A method for diagnosing a bloodcoagulation/anticoagulation disorder or for determining a predispositionthereto in a human, said method comprising: determining anticoagulantFactor V activity in an assay medium containing a sample derived fromsaid human, wherein an abnormal level of the anticoagulant Factor Vactivity indicates manifestation of, or predisposition to, said bloodcoagulation/anticoagulation disorder.
 45. The method of claim 44,wherein the assay medium further comprises a substrate for ActivatedProtein C (APC) and wherein determining anticoagulant Factor V activitycomprises measuring conversion of said substrate by APC and correlatingsaid conversion to said anticoagulant Factor V activity.
 46. The methodof claim 44, wherein said disorder is a thromboembolic disorder.
 47. Themethod of claim 46, wherein said abnormal level is a decreased level.48. The method of claim 44, wherein said anticoagulant Factor V activitycan be correlated to conversion of a substrate specific for activatedProtein C (APC), said method further comprising: measuring in said assaymedium, containing said sample and a substrate for APC, conversion ofsaid substrate by APC and correlating said conversion to saidanticoagulant Factor V activity; wherein at least one exogenoussubstance selected from the group consisting of Protein S and aninhibitor of Protein S activity is added to said assay medium.
 49. Themethod of claim 44, wherein the method further comprises adding to theassay medium a blood coagulation factor or a reagent that activates theblood coagulation system via a pathway selected from the intrinsic andextrinsic pathways.
 50. The method of claim 44, said method furthercomprising adding blood coagulation components selected from the groupconsisting of Factor VII/VIIa, Factor IX, Factor IXa, Factor X/Xa,Factor II, Factor XIa, Factor XIIa and a reagent that serves tointroduce an activated coagulation factor.
 51. The method of claim 50,wherein said reagent that serves to introduce an activated coagulationfactor is selected from the group consisting of a contact activator anda tissue factor.
 52. A method for determining, in a sample derived froma human, a functional activity of anticoagulant Factor V of the humanblood coagulation system, which activity can be correlated to conversionof a substrate specific for activated Protein C (APC), said methodcomprising: measuring in an assay medium containing said sample and asubstrate for APC, conversion of said substrate by APC and correlatingsaid conversion to said functional activity of said anticoagulant FactorV; wherein exogenous APC is added to said assay medium.
 53. The methodof claim 52, wherein said substrate for APC is selected from the groupconsisting of Factor Va and Factor VIIIa.
 54. The method of claim 53,wherein the sample is derived from an individual on therapy with vitaminK antagonists or otherwise deficient in vitamin K-dependent coagulationfactors, and wherein at least one vitamin K dependent coagulation factorin activated or inactivated form is added to the assay medium.
 55. Themethod of claim 52, wherein said method further comprises adding to theassay medium a blood coagulation factor or a reagent that activates theblood coagulation system via a pathway selected from the groupconsisting of the intrinsic and extrinsic pathways.
 56. The method ofclaim 52, wherein said method further comprises adding blood coagulationcomponents selected from the group consisting of Factor VII/VIIa, FactorIX, Factor IXa, Factor X/Xa, Factor II, Factor XIa, Factor XIIa and areagent that serves to introduce an activated coagulation factor. 57.The method of claim 56, wherein said reagent that serves to introduce anactivated coagulation factor is selected from the group consisting of acontact activator and a tissue factor.
 58. The method of claim 52,further comprising a comparison of multiple samples and wherein thefunctional activity of said exogenous APC added to each sample isessentially constant between said samples.
 59. The method of claim 58,wherein a functional excess of said exogenous APC is added to eachsample to achieve said essentially constant functional activity.
 60. Themethod of claim 58, wherein an antibody which specifically binds to anepitope associated with anticoagulant activity of Protein S is added toeach sample to achieve said essentially constant functional activity.61. The method of claim 52, further comprising adding to said assaymedium a substance selected from the group consisting of Factor VIII andVIIIa.
 62. The method of claim 52, wherein said sample is a blood orblood derived sample.
 63. The method of claim 52, wherein saiddetermination of said functional activity is used to diagnose a bloodcoagulation disorder in an individual from which said sample is derived.